Generation of Mature Murine Monocytes from Heterogeneous Bone Marrow and Description of Their Properties

Abstract

Monocytes are involved in a wide range of physiological and pathological processes, many of which are studied in mouse models. Current protocols to isolate murine monocytes are few and result in unsatisfactory cell yield and purity. Here, we describe a novel approach to efficiently differentiate large numbers of mature inflammatory monocytes from heterogeneous bone marrow cell suspensions. Bone marrow cell suspensions were isolated by flushing femurs and tibias from Balb/c and C57Bl/6 mice, supplemented with macrophage colony–stimulating factor (M-CSF), and were cultured on ultra-low attachment surfaces to inhibit adherence-mediated maturation. Cells were harvested at indicated time points, underwent time-line analysis of the differentiation processes, and were subsequently extensively phenotyped to verify their monocytotic properties. In order to confirm downstream compatibility, we tested for typical monocyte behavior. Our protocol yielded 24 ± 6 × 106 differentiated cells per donor mouse, 10-fold higher than yields obtained using previously described peripheral blood isolation methods. Differentiated cells consisted of approximately 47% ± 12% monocytes, the rest being mature macrophages. We increased monocyte purity to 86% ± 6% by depleting adherent macrophages. Our findings indicate that bone marrow–derived monocytes (BMDMs) are an attractive tool to study, for example, the innate and adaptive immune system, atherosclerosis, and cellular migration during infection. Moreover, BMDM transplantation could be used to test novel, therapeutic in vivo approaches in mice disease models.

Physiologically activated mammary fibroblasts promote postpartum mammary cancer

“…Monocyte-derived cells were generated according to previous publications (88,89). Briefly, bone marrow cells were isolated from femurs and tibias of 10-week-old female BALB/c mice, plated in 6-cm Petri dishes at 10 million cells/plate, and cultured in RPMI medium (HyClone, SH30096.01) supplied with 5% FBS, 1× penicillin/streptomycin, and 30% conditioned medium from the supernatants of M-CSFsecreting L929 fibroblasts (90), a gift from Peter Henson (National Jewish Health).…”

TLR7 and TLR9 ligands regulate antigen presentation by macrophages

“…The BMDMs produced by our culture conditions were characterized as Gr-1 neg/low CD64 + , suggesting a mature phenotype (20) (Supplementary Figure S1, available at International Immunology Online). In addition, they expressed CD16/CD32.…”

Tissue damage negatively regulates LPS-induced macrophage necroptosis

“…Bone marrow was flushed out with pre-chilled Dulbecco's Modified Eagle Medium (DMEM) from femurs and tibias, which were harvested from WT and the above-mentioned gene knockout mice following the previously described method. 58,59 In brief, cell pellets were collected by centrifugation at 4°C, and erythrocytes were lysed with RBC lysis buffer (eBioscience, San Diego, CA, USA). The resultant cells were then washed two times with PBS and suspended in BMDM culture medium (DMEM containing 10% fetal bovine serum complemented with 50 μg/ml penicillin/streptomycin and 10 ng/ml recombinant macrophage-colony stimulating factor (M-CSF; Sigma-Aldrich, St Louis, MO, USA)) at a concentration of 10 6 cells/ml and seeded into 6-cm ultra-low Cell staining.…”